Bone marrow transplantation (BMT) is now an important treatment modality for aplastic anemia and leukemia, and BMT strategies are under intense investigation for utility in other malignancies and in genetic disease. Two forms of bone marrow transplantation have been developed, namely, the allogeneic (from a genetically different donor) and autologous (using marrow cryopreserved prior to ablative therapy) forms. Both are based on a principle of high dose chemotherapy and/or radiation therapy followed by repopulation of the marrow by infusion.
Due to the inability to transfer only the stem cell population, the applicability of allogeneic BMT remains restricted by graft vs. host disease (GVHD), which is apparently mediated by T lymphocytes in the graft cell population. Risk of GVHD has limited allogeneic BMT to use only in highly fatal diseases, and even then, only for patients with HLA-matched donors, usually siblings. Autologous BMT can avoid most of the problems associated with allogeneic transplants. In autologous BMT, however, it is necessary to reintroduce only desirable cell populations free of diseased cell populations (e.g., occult tumor cells) to avoid re-introduction of the disease.
The problems associated with both allogeneic and autologous BMT can be alleviated by using purified stem cell populations for the graft. These purified populations can be obtained from marrow cell suspensions by positive selection (collecting only the desired cells) or negative selection (removing the undesirable cells), and the technology for capturing specific cells on affinity materials is well developed. (Wigzel, et al. (1969), J. Exp. Med., 129:23; Schlossman, et al. (1973), J. Immunol., 110:313; Mage, et al. (1977), J. Immunol. Meth., 15:47; Wysocki, et al. (1978), Proc. Nat. Acad. Sci., 75:2844; Schrempf-Decker, et al. (1980), J. Immunol. Meth., 32:285; Muller-Sieburg, et al. (1986), Cell, 44:653.)
Monoclonal antibodies against antigens peculiar to mature, differentiated cells have been used in a variety of "negative" selection strategies to remove undesired cells (i.e., to deplete T cells or malignant cells from allogeneic or autologous marrow grafts, respectively). (Gee, et al., J.N.C.I. (1988) 80:154-9; Gee, al., "Proc. of 1st Int. Workshop on Bone Marrow Purging," in Bone Marrow Transpl., Supp. 2, London, MacMillan, 1987.) Successful purification of human hematopoietic cells by negative selection with monoclonal antibodies and immunomagnetic microspheres has been reported which involved use of multiple monoclonal antibodies, thus making it more costly for clinical application than positive selection. (Griffin, et al., Blood, 63:904 (1984); Kannourakis, et al., Exp. Hematology, 15:1103-1108 (1987).) Most studies report 1 to 2 orders of magnitude reduction in the target cell level following monoclonal antibody treatment. This may not be adequate T lymphocyte depletion necessary to prevent GVHD in allogeneic transplants, and it is certainly insufficient in autologous bone marrow transplantation where 10.sup.6 to 10.sup.9 malignant cells may be present in the patient's marrow.
Positive selection of normal marrow stem cells is an alternative for treatment of the marrow graft. The procedure employs a monoclonal antibody which selectively recognizes human lymphohematopoietic progenitor cells, such as the anti-MY10 monoclonal antibody that recognizes an epitope on the CD34 glycoprotein antigen. Cells expressing the CD34 antigen include essentially all unipotent and multipotent human hematopoietic colony-forming cells (including the pre-colony forming units (pre-CFU) and the colony forming unit-blasts (CFU-Blast)) as well as the very earliest stage of committed B lymphoid cells, but NOT mature B cells, T cells, NK cells, monocytes, granulocytes, platelets, or erythrocytes. See Civin, U.S. Pat. No. 4,714,680.
CFU yields in MY10-positive cell populations are far higher than the 0.1-23% range of recovery of CFU observed after treatment of marrow grafts with 4-hydroperoxycyclophosphamide, a cyclophosphamide metabolite that "purges" malignant cells from marrow grafts without ablating the ability of the marrow to engraft. (Yeager, et al. (1986), N. Eng. J. Med., 315:141.) Positive selection utilizing CD34 monoclonal antibody also appears more feasible (over the long term) for BMT than negative selection strategies for isolations of rare progenitor cells from marrow or blood, offering advantages such as specificity, simplicity, and cost in treatment of diseases other than leukemia.
Recently, Berenson, et al. (1986), J. Immunol. Meth., 91:11-19, disclosed a method for large scale positive selection of class II antigen-positive or CD34-positive cells from marrow, using monoclonal antibody columns. The preliminary results were based on in vitro CFU assays on separated human marrow samples, and actual in vivo BMT experiments in primates. (Berenson, et al. (1988), J. Clin. Invest., 81:951-960.) The primate experiments were possible, since some epitopes of the MY10 glycoprotein are shared between humans and primates.
Marrow cells tend to aggregate nonspecifically at the high cell density that results from slow percolation of marrow through the column necessitated by the relatively low avidity of monoclonal antibody for cell surface antigen, so this work took advantage of the high affinity avidin-biotin interaction. Marrow cells were first labelled with monoclonal antibody, then with biotin-labelled anti-mouse Ig. Upon percolation through a column of avidin-coated macroscopic agarose beads, antigen-positive cells bound to the column, even at high flow rates. After washing of the column to remove unbound cells, bound cells were physically sheared from the beads by vigorous pipetting of the column contents. This release method does not guarantee that all cell-antibody complexes (i.e., antibody-coated cells) were eliminated from the final cell suspension.
Further refinement of techniques for positive selection of MY10-positive cells are available which do not require treatment of marrow cells with multiple reagents (CD34 monocional antibody, biotinylated polyclonal anti-mouse antibody, avidin-conjugated macrobeads). Magnetic microspheres with low nonspecific avidity for cells are commercially available, either in uncoated form (for adsorption of the desired antibody) or coated with anti-mouse Ig. Cell trapping can more readily be avoided with monodisperse microspheres, and the immunomagnetic microsphere technique has been shown to be effective for positive selection in, e.g., Gaudernack, et al., J. Immunol. Meth., 90:179 (1986).
The most desirable cell suspension for BMT would be one that is substantially free of cell:receptor complexes. Thus, the problem of how to release positively selected cells from the affinity matrix once they have been separated from the non-selected cells still remains.